We continued an investigation of the structure of mammalian DNA polymerase Alpha. Our present results emphasize the presence and abundance of a approximately 120,000 Mr-Alpha-polymerase catalytic polypeptide in mouse myeloma cells and in several other mammalian cell types. Catalytic polypeptides of other Mrs also were observed in these cells, and it was found that two molecular forms of purified myeloma Alpha-polymerase had different subunit compositions. Further, we observed that in vitro translation of total calf thymus poly(A+) RNA resulted in synthesis of a 120,000-Mr polypeptide with DNA polymerase activity. A panel of monoclonal antibodies against mammalian DNA polymerase Alpha and Beta were further characterized. Several lines of evidence were obtained that one of these antibodies cross-reacts with Alpha-polymerase and Beta-polymerase. These antibodies are now well enough characterized for use in studies on DNA polymerase structure and gene cloning. Structure-function relationships of E. coli DNA polymerase I large fragment were evaluated using pyridoxal 5'-phosphate covalent modification of the enzyme. Our data indicate that a lysine residue in an essential binding site for dNTP is a target for pyridoxal 5'-phosphate modification.